Is this a Y-adapter design? If so, the P7 and P5 need to anneal over the last 12 bases of the P5/first 12 bases of the P7, with a 3' "T" overhang to work with many Illumina work flows. When you add 8 N's and a T to the P5, you create a 9 base 3' overhang.
No chance you can ligate that to anything using a double stranded DNA ligase.

I suppose the opposite design, where you put the 8 N's at the 5' end of P7 could work. But you would have to anneal the P5 and P7 by their 12 bases of complementarity and then extend the 3' end of the P5 strand using klenow, for example. But that would give you a blunt adapter. So your inserts would need to be blunt as well. Which would allow chimeric inserts and the formation of massive adapter dimers.

to be added to any sequence information derived from their adapter sequences if they are distributed or published outside ones institution.

The correct position to place a UMI in the P5 index site would be after the TCTACAC. That is:
AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT

But, because 8 N's can compose, at most, 4^8 ~= 64K sequence combinations, they would not be "unique" in the context of a sample producing millions of sequences. And, I don't know how far you can push the length of the i5 index. Probably 12, at least. But going beyond 8 bases you would need to subtract sequence from the sequence reads to make up for the reagents you would be using to generate a longer UMI.

1. I am actually not really sure if it is a Y-adapter design. But I am pretty sure that it is. I tried to find information about it but couldn't, but it is the same adapter as used for TruSeq LT.

2. Thank you for suggesting not to go with blunt inserts.

3. Thank you for that suggestion. Do you know if any of the current kits from Illumina uses Dual indexing where you also have the Y-adaptor setup as I presumably have? I would believe that this setup would actually be the best setup to run since I would be sure to have intact annealing at the complementary 12 bases.

Thank you for your answers
Kind regards
Emil

Quote:

Originally Posted by pmiguel

Is this a Y-adapter design? If so, the P7 and P5 need to anneal over the last 12 bases of the P5/first 12 bases of the P7, with a 3' "T" overhang to work with many Illumina work flows. When you add 8 N's and a T to the P5, you create a 9 base 3' overhang.
No chance you can ligate that to anything using a double stranded DNA ligase.

I suppose the opposite design, where you put the 8 N's at the 5' end of P7 could work. But you would have to anneal the P5 and P7 by their 12 bases of complementarity and then extend the 3' end of the P5 strand using klenow, for example. But that would give you a blunt adapter. So your inserts would need to be blunt as well. Which would allow chimeric inserts and the formation of massive adapter dimers.

to be added to any sequence information derived from their adapter sequences if they are distributed or published outside ones institution.

The correct position to place a UMI in the P5 index site would be after the TCTACAC. That is:
AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT

But, because 8 N's can compose, at most, 4^8 ~= 64K sequence combinations, they would not be "unique" in the context of a sample producing millions of sequences. And, I don't know how far you can push the length of the i5 index. Probably 12, at least. But going beyond 8 bases you would need to subtract sequence from the sequence reads to make up for the reagents you would be using to generate a longer UMI.

Hi Emil,
Most TruSeq Illumina kits use the Y-adapters. The common "TruSeq" DNA and RNAseq ones will offer a normal single index kit option (usually going up to 24 indexes) or a "high thoughput" one with dual indexes allowing you to multiplex 96 samples in a lane. Well, as long as you don't need them to be "unique dual".

The parts of the dual index Y-adapters containing the indexes do not anneal! Only the 12 bases of the Y-adapter most proximate to the insert are annealled. The rest of the adapter has non-complementary sequence that won't anneal and hangs off like a forked tail. Get it? "Y" where the double-stranded part is the stem of the "Y" and the single stranded tails are the top part. It is only during subsequent PCR that this tail region becomes double stranded.

This is one of those brilliant solutions to a bunch amplicon construction issues when using ligation. You need to have a forward and a reverse adapter. But if you just make them both double-stranded, so that T4 DNA ligase will use them as a substrate, you create 2 problems. First some of your constructs will get forward adapters on both ends or reverse adapters on both end and won't be suitable templates for clustering. Second with a double-stranded molecule you have two ends that can be ligated -- possibly to each other.

So some fiendish genius came up with the idea of annealing single-stranded versions of both the left and right adapters to each other such that only one end was actually annealed. This way each double stranded insert was guaranteed to have an R adapter on one end and an F adapter on the other end. Actually, each strand of the the double stranded insert would have a single stranded R on one end and F on the other--the top strand in one orientation (say, F-insert-R) the bottom strand in the other orientation (R-insert-F).

It has 6 UMI base which follows the index read so the index 1 read has to be 12 cycles to utilize UMI or 6 cycles just for the index. Other advantage is that they have included diversity nucleotides and libraries can be sequenced with 1% PhiX spike in. In the conventional protocol higher PhiX (>30%) is required.

I have allready generated the TruSeq DNA LT adapter piece with a 6 nt index. Do you think it would work to anneal the i5 adapter to this adapter or should i generate new i7 adapters aswell?

Also to nucacidhunter and torben, thanks for the suggestions!

Cheers
Emil

Quote:

Originally Posted by pmiguel

Hi Emil,
Most TruSeq Illumina kits use the Y-adapters. The common "TruSeq" DNA and RNAseq ones will offer a normal single index kit option (usually going up to 24 indexes) or a "high thoughput" one with dual indexes allowing you to multiplex 96 samples in a lane. Well, as long as you don't need them to be "unique dual".

The parts of the dual index Y-adapters containing the indexes do not anneal! Only the 12 bases of the Y-adapter most proximate to the insert are annealled. The rest of the adapter has non-complementary sequence that won't anneal and hangs off like a forked tail. Get it? "Y" where the double-stranded part is the stem of the "Y" and the single stranded tails are the top part. It is only during subsequent PCR that this tail region becomes double stranded.

This is one of those brilliant solutions to a bunch amplicon construction issues when using ligation. You need to have a forward and a reverse adapter. But if you just make them both double-stranded, so that T4 DNA ligase will use them as a substrate, you create 2 problems. First some of your constructs will get forward adapters on both ends or reverse adapters on both end and won't be suitable templates for clustering. Second with a double-stranded molecule you have two ends that can be ligated -- possibly to each other.

So some fiendish genius came up with the idea of annealing single-stranded versions of both the left and right adapters to each other such that only one end was actually annealed. This way each double stranded insert was guaranteed to have an R adapter on one end and an F adapter on the other end. Actually, each strand of the the double stranded insert would have a single stranded R on one end and F on the other--the top strand in one orientation (say, F-insert-R) the bottom strand in the other orientation (R-insert-F).

Hi Emil,
I would strongly recommend that you verify this yourself by aligning your p5 and the reverse (in the 3' - 5' direction) of your p7 sequence. You will see the terminal 12 bases on one side are complements of each other with just a 3' "T" overhang provided by the p5 oligo.
Once you have done that, you will understand how a Y-adapter is structured to function as it does.
--
Phillip

I hope that you have asked all C residues to be synthesized with mC to prevent C conversion to U during bisulfite treatment (which is very expensive) unless you are using techniques that does not require mC in adapters.